Determination of Hematocrit (Hct) / Packed Cell Volume (PCV)

Introduction

The hematocrit (Hct), also known as packed cell volume (PCV), is a crucial diagnostic test in hematology. It measures the percentage of the total blood volume that consists of red blood cells (RBCs). This simple yet vital parameter provides immediate information regarding the oxygen-carrying capacity of the blood and is essential in diagnosing and managing conditions such as anemia and polycythemia.

Because hematocrit is directly related to the red blood cell count and the mean corpuscular volume (MCV), it serves as a key component of the Complete Blood Count (CBC). In clinical practice, the hematocrit is often one of the first tests ordered when a patient presents with symptoms of fatigue, pallor, or shortness of breath.

Definition and Principle

The term "hematocrit" is derived from the Greek words "hema" (blood) and "krites" (judge). Historically, it referred to the separation of blood components. The principle behind the determination of hematocrit is based on the differential density of blood cells and plasma.

When an anticoagulated blood sample is centrifuged at high speeds, the heavier red blood cells are forced to settle at the bottom of the tube. Above the RBCs, a thin whitish layer known as the "buffy coat" forms, containing white blood cells (WBCs) and platelets. The top layer consists of plasma. The hematocrit is calculated by measuring the length of the column of packed red blood cells relative to the total length of the blood column in the tube.

Methods of Determination

There are primarily two methods used to determine hematocrit: the Macro method (Wintrobe method) and the Micro method (Microhematocrit method). While electronic cell counters provide automated readings in modern labs, the manual microhematocrit method remains the "gold standard" for reference due to its accuracy and cost-effectiveness.

The Macro Method (Wintrobe Method)

This traditional method utilizes a Wintrobe hematocrit tube, which is a thick glass tube approximately 11 cm long with a uniform bore. The tube is graduated from 0 to 10 at the bottom and 10 to 0 at the top.

• Blood is drawn into a capillary tube or directly from the patient via venipuncture into an anticoagulant tube.
• The Wintrobe tube is filled with blood to the "0" mark using a long-necked Pasteur pipette.
• The tube is centrifuged at 3,000 rpm for 30 minutes.
• After centrifugation, the reading is taken at the top of the red cell column.

While accurate, this method requires a larger volume of blood and is more time-consuming than the micro method.

The Micro Method (Microhematocrit)

This is the most widely used manual technique. It requires only a small drop of blood, making it ideal for pediatric patients, fingerstick samples, or situations where blood volume is limited.

• Capillary tubes (typically 75 mm long and 1 mm diameter) coated with heparin (an anticoagulant) are used.
• A capillary tube is filled approximately 2/3 to 3/4 full with blood by capillary action.
• One end of the tube is sealed using a sealing clay or a flame.
• The tubes are placed in a microhematocrit centrifuge with the sealed end facing outward.
• The samples are centrifuged for 5 minutes at 10,000 to 15,000 rpm.
• Using a hematocrit reader or a simple ruler, the packed cell volume is determined.

Clinical Procedure (Micro Method)

To ensure accurate results, the procedure must be followed with precision. Fingerstick or venous blood can be used. If using fingerstick, ensuring adequate blood flow is critical to avoid the need for re-puncture.

1. Clean the puncture site (finger or earlobe) with 70% alcohol and allow it to dry.
2. Puncture the skin with a sterile lancet. Wipe away the first drop of blood to avoid tissue fluid contamination.
3. Touch the open end of the heparinized capillary tube to the second drop of blood. The blood will flow into the tube by capillary action. Ensure there are no air bubbles.
4. Fill the tube to at least the 50% mark, ideally 3/4 full.
5. Seal the unfilled end of the tube safely using a plastic sealant or clay. Avoid heating the blood if using flame sealing, as this can hemolyze the cells.
6. Place the tubes in the centrifuge rotor, ensuring the sealed ends point outward.
7. Centrifuge for the standard time (usually 5 minutes).
8. Remove the tube and place it in a hematocrit reader.
9. Move the zero line on the reader to the top of the plasma (meniscus) and the 100% line to the bottom of the red cell column.
10. Read the value at the top of the red cell column.

Reference Values

Normal hematocrit values vary depending on age, sex, and physiological conditions. Generally, males have higher values than females due to the effect of androgens stimulating erythropoiesis.

• Adult Men: 41% to 53%
• Adult Women: 36% to 46%
• Newborns: 47% to 67% (Values are high at birth and decrease over the first few months)
• Children (1 year): 32% to 40%

Clinical Significance

Variations in hematocrit levels indicate pathology or physiological changes within the body. The Hematocrit is interpreted in conjunction with Hemoglobin (Hb) and RBC indices.

Increased Hematocrit (Polycythemia)

An elevated hematocrit indicates an excess of red blood cells. This can be relative (due to decreased plasma volume) or absolute (due to increased RBC production).

• Dehydration: Loss of fluid (burns, diarrhea, vomiting) reduces plasma volume, artificially raising the percentage of RBCs.
• Polycythemia Vera: A myeloproliferative disorder where the bone marrow produces excessive cells.
• Chronic Hypoxia: Living at high altitudes or smoking stimulates erythropoietin production to increase oxygen delivery.
• Renal Tumors: Tumors may secrete erythropoietin, causing excessive RBC formation.

Decreased Hematocrit (Anemia)

A low hematocrit signifies a reduction in the mass of red blood cells. The condition results in reduced oxygen delivery to tissues.

• Nutritional Deficiencies: Lack of Iron, Vitamin B12, or Folic acid impairs RBC synthesis.
• Hemorrhage: Acute or chronic blood loss reduces RBC count.
• Hemolysis: Destruction of RBCs (e.g., autoimmune hemolytic anemia, transfusion reactions) lowers the hematocrit.
• Bone Marrow Disorders: Aplastic anemia or leukemia where the marrow fails to produce cells.
• Chronic Kidney Disease: Decreased production of erythropoietin.

Sources of Error

Determining hematocrit is relatively simple, but pre-analytical and technical errors can affect the result.

• Improper Mixing: If blood is collected in a tube with anticoagulant, it must be mixed gently. Failure to mix causes clotting.
• Incorrect Centrifugation: Insufficient speed or time results in trapped plasma between RBCs, artificially increasing the hematocrit reading.
• Increased Buffy Coat: In conditions like leukemia, the buffy coat layer expands significantly, mimicking RBC volume.
• Capping: Incorrect sealing can lead to leakage of blood during centrifugation.
• Venous Occlusion: Leaving the tourniquet on too long during venipuncture causes hemoconcentration (RBCs shift into the vessel), leading to falsely high values.

Calculation

When using a manual reader, the calculation is direct:

Hematocrit (%) = (Length of Packed RBCs / Total Length of Blood Column) x 100

It is important to note that automated estimations of hematocrit are "calculated" values. Most automated cell counters calculate hematocrit by multiplying the RBC count by the Mean Corpuscular Volume (MCV) and dividing by 10:

Hct = RBC count (mill/L) x MCV (fL) / 10

Conclusion

The determination of Hematocrit (PCV) remains one of the most fundamental procedures in medical laboratory science. Despite the advent of sophisticated automated analyzers, the microhematocrit method is favored for its speed, simplicity, and reliability. It provides critical data for the rapid assessment of a patients oxygen-carrying capacity, hematological status, and hydration levels. Understanding the principles, procedure, and potential pitfalls ensures diagnostic accuracy, facilitating better patient management and care.